Current eye research · 1997
Cytoplasmic Ca2+ mobilization and Ca(2+)-dependent membrane currents in dispersed bovine ciliary muscle cells.
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Fujii T, Tokutomi N, Hirata A, Negi A, Nishi K
Аннотация
PURPOSE: The dependence of plasmalemma Ca2+ influx and Ca2+ release from intracellular stores on Ca2+ activated K+ channels of bovine ciliary muscle (CM) cells were examined.
METHODS: The nystatin-perforated patch clamp technique for the measurement of membrane currents and a microscope based fura-2 fluorescence imaging of [Ca2+]i were applied to CM cells freshly dissociated with collagenase and identified with smooth muscle-specific alpha-isoactin.
RESULTS: At holding voltages (VH) of > -60 mV, CM cells showed spontaneous transient outward currents (STOCs) and caffeine (> 10(-4) M) induced large transient outward currents (ICAF). Both STOCs and ICAF were abolished by tetraethylammonium chloride (10(-3) M) and charybdotoxin (10(-7) M), but not by apamin (10(-6) M), suggesting that both currents are mediated by Ca(2+)-activated K+ channels similar to those with medium (MK) or large (BK-type) conductance. Both STOCs and ICAF were gradually abolished in the nominally Ca(2+)-free and Co(2+)-containing solutions but were resistant to L-type Ca2+ channel blockers, including nicardipine, verapamil and diltiazem and a N-type channel blocker, omega-contoxin. The [Ca2+]i-elevation during high K+ (100 mM)-depolarization was prevented by Ca(2+)-free and Co(2+)-containing solutions but not by nicardipine.
CONCLUSIONS: These results suggest that CM cells possess MK or BK type-like Ca(2+)-activated K+ channels and that L-type Ca2+ channels play minor roles for the maintenance of Ca(2+)-dependent responses in contrast to other types of smooth muscle cells.
METHODS: The nystatin-perforated patch clamp technique for the measurement of membrane currents and a microscope based fura-2 fluorescence imaging of [Ca2+]i were applied to CM cells freshly dissociated with collagenase and identified with smooth muscle-specific alpha-isoactin.
RESULTS: At holding voltages (VH) of > -60 mV, CM cells showed spontaneous transient outward currents (STOCs) and caffeine (> 10(-4) M) induced large transient outward currents (ICAF). Both STOCs and ICAF were abolished by tetraethylammonium chloride (10(-3) M) and charybdotoxin (10(-7) M), but not by apamin (10(-6) M), suggesting that both currents are mediated by Ca(2+)-activated K+ channels similar to those with medium (MK) or large (BK-type) conductance. Both STOCs and ICAF were gradually abolished in the nominally Ca(2+)-free and Co(2+)-containing solutions but were resistant to L-type Ca2+ channel blockers, including nicardipine, verapamil and diltiazem and a N-type channel blocker, omega-contoxin. The [Ca2+]i-elevation during high K+ (100 mM)-depolarization was prevented by Ca(2+)-free and Co(2+)-containing solutions but not by nicardipine.
CONCLUSIONS: These results suggest that CM cells possess MK or BK type-like Ca(2+)-activated K+ channels and that L-type Ca2+ channels play minor roles for the maintenance of Ca(2+)-dependent responses in contrast to other types of smooth muscle cells.